The Role of HIF-1α-Coactivator Interactions in Regulation of VEGF Transcription
The interaction between the cysteine-histidine rich 1 domain (“CH1”) of the coactivator protein p300 (or the homologous CREB binding protein, CBP) and the C-terminal transactivation domain (“C-TAD,” aa 786-826 of NCBI accession number NP 001521) of the hypoxia-inducible factor 1α (“HIF-1α”) (Freedman et al., “Structural Basis for Recruitment of CBP/p300 by Hypoxia-inducible Factor-1α,” Proc. Nat'l Acad. Sci. USA 99:5367-72 (2002); Dames et al., “Structural Basis for HIF-1α/CBP Recognition in the Cellular Hypoxic Response,” Proc. Nat'l Acad. Sci. USA 99:5271-6 (2002)) mediates transactivation of hypoxia-inducible genes (Hirota & Semenza, “Regulation of Angiogenesis by Hypoxia-inducible Factor 1,” Crit. Rev. Oncol. Hematol. 59:15-26 (2006); Semenza, “Targeting HIF-1 for Cancer Therapy,” Nat. Rev. Cancer 3:721-32 (2003)). Hypoxia-inducible genes are important contributors in angiogenesis and cancer metastasis, as shown in FIGS. 1A-C (Orourke et al., “Identification of Hypoxically Inducible mRNAs in HeLa Cells Using Differential-display PCR,” Eu. J. Biochem. 241:403-10 (1996); Ivan et al., “HIFα Targeted for VHL-mediated Destruction by Proline Hydroxylation: Implications for O2Sensing,” Science 292:464-8 (2001)). Under normoxia, the α-subunit of HIF-1 is successively hydroxylated at proline residues 402 and 564 by proline hydroxylases, ubiquitinated, and then degraded by the ubiquitin-proteosome system, as shown in FIG. 2. This process, mediated by the von Hippel-Lindau tumor suppressor protein (Kaelin, “Molecular Basis of the VHL Hereditary Cancer Syndrome,” Nat. Rev. Cancer 2:673-82 (2002)), is responsible for controlling levels of HIF-1α and, as a result, the transcriptional response to hypoxia (Maxwell et al., “The Tumour Suppressor Protein VHL Targets Hypoxia-inducible Factors for Oxygen-dependent Proteolysis,” Nature 399:271-5 (1999)). Under hypoxic conditions, HIF-1α is no longer targeted for destruction and accumulates. Heterodimerization with its constitutively expressed binding partner, aryl hydrocarbon receptor nuclear translocator (“ARNT”) (Wood et al., “The Role of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) in Hypoxic Induction of Gene Expression,” J. Biol. Chem. 271: 15117-23 (1996)) results in binding to a cognate hypoxia response element (“HRE”) (Forsythe et al., “Activation of Vascular Endothelial Growth Factor Gene Transcription by Hypoxia-inducible Factor 1,” Mol. Cell. Biol. 16:4604-13 (1996)). A third site of regulatory hydroxylation on Asparagine 803 is also inhibited under hypoxic conditions (Lando et al., “FIH-I is an Asparaginyl Hydroxylase Enzyme That Regulates the Transcriptional Activity of Hypoxia-inducible Factor,” Genes & Develop. 16:1466-71 (2002)), allowing recruitment of the p300/CBP coactivators, which trigger overexpression of hypoxia inducible genes, as shown in FIG. 2. Among these are genes encoding angiogenic peptides such as vascular endothelial growth factor (“VEGF”) and VEGF receptors VEGFR-I (Flt-1) and VEGFR-2 (KDR/Flk-1), as well as proteins involved in altered energy metabolism, such as the glucose transporters GLUT1 and GLUT3, and hexokinases 1 and 2 (Forsythe et al., “Activation of Vascular Endothelial Growth Factor Gene Transcription by Hypoxia-inducible Factor 1,” Mol. Cell. Biol. 16:4604-13 (1996); Okino et al., “Hypoxia-inducible Mammalian Gene Expression Analyzed in Vivo at a TATA-driven Promoter and at an Initiator-driven Promoter,” J. Biol. Chem. 273:23837-43 (1998)).
Epidithiodiketopiperazine Fungal Metabolites as Regulators of Hypoxia-inducible Transcription
Because interaction of HIF-1α C-TAD with transcriptional coactivator p300/CBP is a point of significant amplification in transcriptional response, its disruption with designed protein ligands could be an effective means of suppressing aerobic glycolysis and angiogenesis (i.e., the formation of new blood vessels) in cancers (Hirota & Semenza, “Regulation of Angiogenesis by Hypoxia-inducible Factor 1,” Crit. Rev. Oncol. Hematol. 59:15-26 (2006); Ramanathan et al., “Perturbational Profiling of a Cell-line Model of Tumorigenesis by Using Metabolic Measurements,” Proc. Nat'l Acad. Sci. USA 102:5992-7 (2005); Underiner et al., “Development of Vascular Endothelial Growth Factor Receptor (VEGFR) Kinase Inhibitors as Anti-angiogenic Agents in Cancer Therapy,” Curr. Med. Chem. 11:73145 (2004)). Although the contact surface of the HIF-1α C-TAD with p300/CBP is extensive (3393 Å2) the inhibition of this protein-protein interaction may not require direct interference. Instead, the induction of a structural change to one of the binding partners (p300/CBP) may be sufficient to disrupt the complex (Kung et al., “Small Molecule Blockade of Transcriptional Coactivation of the Hypoxia-inducible Factor Pathway,” Cancer Cell 6:33-43 (2004)).
Although inhibition of nuclear protein-protein interactions with small molecules in the past has proven to be difficult (Arkin & Wells, “Small-molecule Inhibitors of Protein-Protein Interactions: Progressing Towards the Dream,” Nat. Rev. Drug Discov. 3:301-17 (2004)), recent screens for high-affinity protein ligands have resulted in several remarkable accomplishments (Kung et al., “Small Molecule Blockade of Transcriptional Coactivation of the Hypoxia-inducible Factor Pathway,” Cancer Cell 6:33-43 (2004); Issaeva et al., “Small Molecule RITA Binds to p53, Blocks p53-HDM-2 Interaction and Activates p53 Function in Tumors,” Nat. Med. 10: 1321-8 (2004); Lepourcelet et al., “Small-molecule Antagonists of the Oncogenic Tcf/β-Catenin Protein Complex,” Cancer Cell 5:91-102 (2004); Vassilev et al., “In Vivo Activation of the p53 Pathway by Small-molecule Antagonists of MDM2,” Science 303:844-8 (2004); Grasberger et al., “Discovery and Cocrystal Structure of Benzodiazepinedione HDM2 Antagonists That Activate p53 in Cells,” J. Med. Chem. 48:909-12 (2005); Ding et al., “Structure-based Design of Potent Non-peptide MDM2 Inhibitors,” J. Am. Chem. Soc. 127:10130-1 (2005); Berg et al., “Small-molecule Antagonists of Myc/Max Dimerization Inhibit Myc-induced Transformation of Chicken Embryo Fibroblasts,” Proc. Nat'l Acad. Sci. USA 99:3830-5 (2002); International Patent Publication No. WO 2006/066775 to De Munari et al.). Two small molecules, chaetocin 1 (Hauser et al., “Isolation and Structure Elucidation of Chaetocin,” Hely. Chirn. Acta 53(5):1061-73 (1970)) (shown in FIG. 3A) and chetomin 2 (Waksman & Bugie, “Chaetomin, a New Antibiotic Substance Produced by Chaetomium Cochliodes I. Formation and Properties,” J. Bacteriol. 48:527-30 (1944)) (shown in FIG. 3B), have been shown to inhibit the interaction between HIF-1α C-TAD and p300/CBP and to attenuate hypoxia-inducible transcription, although the exact mechanism of this inhibition remains unclear (Kung et al., “Small Molecule Blockade of Transcriptional Coactivation of the Hypoxia-inducible Factor Pathway,” Cancer Cell 6:33-43 (2004)). Despite the initial encouraging reports, further design of inhibitors of the HIF-1 pathway is needed, because both 1 and 2 have induced coagulative necrosis, anemia, and leukocytosis in experimental animals. It would be desirable to identify other inhibitors of the HIF-1 pathway that lack or have diminished side effects.
The present invention is directed to overcoming these and other deficiencies in the art.